Gene Expression and Methylation Pattern in HRK Apoptotic Gene in Myelodysplastic Syndrome.

Myelodysplastic syndromes (MDSs) are a clonal bone marrow (BM) disease characterized by ineffective hematopoiesis, dysplastic maturation and progression to acute myeloid leukemia (AML). Methylation silencing of HRK has been found in several human malignancies. In this study, we explored the association of HRK methylation status with its expression, clinical parameters and MDS subtypes in MDS patients. To study the methylation status of HRK gene, we applied Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) in MDS patients, as well as healthy controls and EpiTect®PCR Control DNA. Real time RT-PCR was used for gene expression analysis. Methylation frequency in promoter region of HRK in patient samples was 20.37%. Methylation of HRK was significantly related to transcriptional downregulation (P=0.023). The difference in frequency of hypermethylated HRK gene was significant between good (10%) and poor (71.42%) cytogenetic risk groups (P= 0.001), advanced stage MDS patients (66.66%) in comparison with early stage MDS patients (2.56%) (P= 0.00), higher- risk MDS group (61.53%) and lower- risk MDS group (7.31%) (P= 0.00). HRK hypermethylation was associated with advanced- stage MDS and downregulation of HRK gene may play a role in the progression of MDS.


Bisulfite conversion
Genomic DNA isolation was performed using QIAamp

Gene expression analysis
The expression level of HRK was validated by

Statistical analyzes
Statistical analyses were performed using SPSS 16.0 software package (SPSS, Chicago, IL).

Mann-Whitney's U-test and T-test were performed
to compare variables between patient and control groups. Kruskal-Wallis test was used to recognize the differences in methylation between subgroups.
ANOVA and student's t-test were performed to evaluate parametric data between different groups.
Chi square was utilized as required. For all analyses, the p values were two-tailed, and a p≤0.05 was considered as statistically significant.  Clinical and demographic data are summarized in Table 1.

HRK mRNA expression in MDS patients
QRT-PCR was carried out to specify whether the methylation in transcription start site was associated with suppression of mRNA expression.
When the delta Ct of HRK was compared between patient and normal groups, the difference was not statistically significant, due to widespread delta Ct    Increase in LDH level (P=0.006), age (P=0.016) and blast count (P=0.00) was observed in high-risk group, which was significantly higher compared to low-risk group. There was significant correlation between IPSS-R prognostic risk categories and Hb, ANC, Plt count and SF and LDH levels (P<0.05).
The LDH level was significantly higher in patients with >5% blasts than group with <5% blasts (P<0.01). There was no difference in SF and LDH levels and hematologic parameters between patients with normal and abnormal karyotype. examined (19). Abnormal methylation of HRK promoter prevented binding of AP-2α and resulted in down regulation of HRK expression, which was followed by resistance to apoptosis and enhanced tumor growth (15). Therefore, HRK might be used as a target for demethylation therapy to stimulate apoptosis in cancer cells (19).

Discussion
Using showed that a serum LDH activity of ≥300 U/L in MDS is associated with a significantly shorter survival and higher risk of AML transformation (27)(28). In addition, it has been shown that a high SF at diagnosis is a poor prognostic factor for overall survival in MDS and a baseline SF level could be an indicator for prediction of leukemic evolution in MDS (29). In our analysis, higher-risk MDS was significantly associated with higher SF level.
In summary, the data reported here show that HRK proapoptotic gene is suppressed by aberrant methylation of its promoter. We further suggest that patients with HRK methylation have a higher incidence of poor karyotype, advanced-stage MDS and higher IPSS-R score than patients without methylation. Defective expression of HRK may contribute to progression of MDS. Moreover, the data presented here indicate that LDH and SF are both significant prognostic factors that may be used for evaluation of prognosis in MDS patients.